The present invention relates to a method of recovering nucleic acids and an apparatus thereof, more particularly a method and apparatus fit to recover nucleic acids from some kinds of sample substances without reducing the concentrations of the nucleic acids.
As the molecular biology advances, many kinds of gene-related technologies have been developed and used to isolate and identify various infected genes. As the result, these molecular biological techniques have been employed by a wide variety of fields including medical, diagnostic, and testing fields, enabling various new diagnostic methods that had not been realized by conventional methods and dramatically shortening test periods.
This rapid progress is attributed mainly by a nucleic acid amplifying method, particularly by PCR (Polymerase Chain Reaction).
As PCR can peculiarly amplify nucleic segments of a specific structure in a solution, for example, PCR can be used to testify to the existence of an extremely small amount of viruses in blood indirectly by amplifying and detecting a nucleic acid which is the gene of the virus.
However, PCR has some problems when it is used for daily testing jobs in a clinical field. The main concern is extraction and refining of nucleic acids in preprocessing. These steps are greatly affected by the inhibiting factors which remain unremoved after refining. Hemoglobin in blood and surface-active agent used for extraction have been widely known as such inhibiting factors.
Further, the extraction process requires time- and labor-consuming complicated manual operations by experts, which mainly causes a hospital to hesitate to employ a new gene testing system. The automation of this process has been longed for.
In a blood center which must quickly detect HCVs (hepatitis C virus), HIV (human immunodeficiency virus), and so on in a great many blood specimens, a screening (sieving) method is sometimes employed to quicken the test. The screening method comprises steps of mixing some specimens into one and testing it. This is because such viruses (HCV, HIV, etc.) are rarely detected and most specimen mixtures are free from such viruses. Only when such viruses are detected in a specimen mixture, the specimens of the specimen mixture are individually tested.
However, in this screening method, the specimen mixture is diluted too much for example, one fiftieth when 50 specimens are combined into one or one five-hundredth when ten 50-specimen mixtures are combined into one and the concentrations of specimens may go below those required to detect nucleic acids which are the genes of such viruses. In extreme cases, the virus-positive specimens may be evaluated as virus-negative.
For extraction of nucleic acids, Japanese Non-examined Patent Publication H08-320274 (1996) discloses a method of isolating genes by means of a plurality of containers and tips for a single specimen. This method comprises the steps of mounting a first tip onto the pipette nozzle which is moved by a driving mechanism, sucking up a specimen into the first tip, fitting a filter which breaks blood corpuscles to the lower end of the first tip and discharging the specimen from the first tip to a first container through this filter.
The method further comprises the steps of demounting the filter and the first tip from the pipette nozzle, mounting a second tip to the lower end of the second tip, and sucking the specimen from the first container into the second tip.
The method furthermore comprises the steps of fitting a silica membrane filter to the lower end of the second tip to catch genes, and discharging the specimen from the second tip to the second container through the silica membrane filter. With this, genes are caught by the silica membrane and impurities are discharged to the second container.
Further, the method comprises the steps of moving the pipette nozzle to a third container which contains a washing liquid, demounting the silica membrane filter which has genes from the second tip, immersing the filter into the washing liquid in the third container, mounting the third tip to the pipette nozzle from which the second tip was demounted, fitting the washed silica membrane to the lower end of the third tip, sucking up a mixture of the washing liquid and the genes into the third tip, and discharging the mixture to the fourth container.
Japanese Non-examined Patent Publication H02-289596 (1990) discloses a method of using silica particles which can bind with nucleic acids in the presence of a chaotropic agent as a stationary phase for binding nucleic acids. This method comprises the steps of adding a specimen containing nucleic acids to a reaction container which contains a silica particle suspension and guanizithiocyanate buffer solution working as the chaotropic agent, mixing thereof, centrifugally separating a complex which binds nucleic acids to silica particles, and disposing of the supernatant solution.
This method further comprises the steps of adding a washing liquid to the complex residue, washing thereof by means of a vortex mixer, washing the complex precipitate with an aqueous solution of ethanol, washing the precipitate again with acetone, removing acetone, drying the precipitate, adding a buffer solution for elution to the dried complex, and recovering the eluted nucleic acids.
Further, Japanese Non-examined Patent Publication H11-266864 (1999) discloses a method comprising the steps of connecting a nucleic acid capturing tip which contains a silica stationary phase to a nozzle, sucking and discharging a mixture of a specimen which contains nucleic acids and a substance which accelerates the nucleic acids to be bound to the stationary phase, causing the nucleic acids to be bound to the stationary phase in the nucleic acid capturing tip, and washing this tip.
The technology in accordance with Japanese Non-examined Patent Publication H11-266864 (1999) enables automated extraction of nucleic acids.
However, as the technology in accordance with Japanese Non-examined Patent Publication H08-320274 (1996) is so constructed to capture genes when discharging the specimen from the second tip through the silica membrane, the time in which the specimen is in contact with the silica membrane is very short and the rate of capturing genes is low. This may cause a suspected virus-negative case.
Further, the technology in accordance with Japanese Non-examined Patent Publication H02-289596 (1990) requires a centrifugal separation process which is an obstacle to automation of the refining process and takes a lot of time for refining.
As already explained above, although the screening method can increase the test speed, the specimen is diluted down to about one hundredth and high-accuracy detection of viruses (HCV, HIV, etc.) cannot be expected.
Therefore, any other fast and high-precision testing method than the screening method has been longed for.
A comprehensive object of the present invention is to provide a method and apparatus which can automatically recover nucleic segments of a specific structure from a bio specimen.
The present invention has attained the aforesaid object by Claims, particularly by making a plurality of specimens in contact with a stationary phase for extracting nucleic acids, capturing nucleic acids from the specimens by a single stationary phase, and extracting thereof by an eluate.
A first embodiment of the present invention comprises the steps of making a plurality of specimens containing nucleic components in contact with a stationary phase which can bind with said nucleic components, letting said stationary phase absorb the nucleic acids from the specimens, separating other components which are left unabsorbed from said stationary phase, performing said two processes on each of the other specimens, letting said stationary phase absorb nucleic acids, making an eluate in contact with said stationary phase, discharging thereof, and eluting. This embodiment further comprises the steps of removing components that are not absorbed by the stationary phase, applying a washing liquid to said stationary phase, and discharging the washing liquid to wash the stationary phase.
A second embodiment of the present invention comprises the steps of passing a plurality of specimens containing nucleic components into a capillary having a stationary phase which can bind with said nucleic components, letting said stationary phase absorb and bind with the nucleic acids in the specimen, discharging the other components that are not absorbed and bound by said stationary phase, washing the stationary phase with a washing liquid by sucking the washing liquid into the capillary and discharging the washing liquid into the capillary, repeating said steps on every specimen to cause the identical stationary phase to absorb and bind with the nucleic acids, sucking an eluate into said capillary, discharging the eluate, and thus eluting the nucleic components from the stationary phase. This method can have a step of adding a substance which accelerates binding of the nucleic components having a specific structure with the stationary phase to said specimen before the step of passing a plurality of specimens containing nucleic components into a capillary having a stationary phase which can bind with said nucleic components and letting said stationary phase absorb and bind with the nucleic acids in the specimen. Further, a specimen containing nucleic components can be divided into some samples. Said capillary should have a capillary tip at the end thereof and said stationary phase can be placed in said tip. Further said tip should be mountable on and demountable from said capillary.
A third embodiment of the present invention comprises the steps of preparing a plurality of specimens N (wherein xe2x80x9cNxe2x80x9d is the number of specimens equal to or greater than 10) containing nucleic components, mixing at least one part of said specimens, preparing mixed specimens N/n (wherein xe2x80x9cnxe2x80x9d is an integer equal to or greater than 2 and xe2x80x9cnxe2x80x9d of respective mixed specimens can be identical or different) or a set of mixed specimens and single specimens, making said specimen (mixed or single) in contact with a stationary phase which can bind with said nucleic components, making said stationary phase contact with and absorb the nucleic acids in the specimens, removing the other components which are not absorbed and bound from said stationary phase, feeding a washing liquid to said stationary phase, discharging said washing liquid (thus washing the stationary phase), repeating these steps on each of the other mixed or single specimens, letting the same stationary phase absorb and bind with nucleic components, supplying an eluate to the stationary phase, discharging the eluate from the stationary phase, and thus eluting the nucleic components from said stationary phase. In other words, an object of the present invention is to recover nucleic acids from a lot of specimens or samples without reducing the concentrations of the nucleic acids. If necessary or if circumstances permit, the method in accordance with the present invention can comprise the steps of mixing a plurality of specimens into one sample, performing absorption, binding, and washing steps on this sample (mixture of specimens), repeating these steps on the other mixed or single sample, letting the same stationary phase to absorb nucleic components, and finally eluting nucleic acids from the stationary phase. Further, this method can have a step of adding a substance which accelerates binding of the nucleic components having a specific structure with the stationary phase to said specimen before the step of passing said specimens through the stationary phase. This step can let the stationary phase absorb and bind nucleic acids of the selected structure only.
A fourth embodiment of the present invention comprises the steps of
performing a first process which comprises the steps of mixing a plurality of prepared specimens N (wherein xe2x80x9cNxe2x80x9d is the number of specimens equal to or greater than 10) containing nucleic components, preparing mixed specimens N/n (wherein xe2x80x9cnxe2x80x9d is an integer equal to or greater than 2 but smaller than xe2x80x9cNxe2x80x9d and xe2x80x9cnxe2x80x9d of respective mixed specimens can be identical or different) or a set of mixed specimens and single specimens, making said specimen (mixed or single) in contact with a stationary phase which can bind with nucleic components of a specific structure in a container, and letting said stationary phase absorb and bind with said nucleic acids in the specimen and a second process which comprises a step of removing the other components which are not absorbed and bound from said stationary phase on each of the other mixed or single specimens,
letting said stationary phase absorb and bind with the target nucleic acids,
supplying an eluate into said container,
discharging the eluate from the stationary phase, and
thus eluting the nucleic components from said stationary phase.
A fifth embodiment of the present invention comprises the steps of
performing a first process which comprises the steps of mixing a plurality of prepared specimens N (wherein xe2x80x9cNxe2x80x9d is the number of specimens) containing nucleic components, preparing mixed specimens N/n (wherein xe2x80x9cnxe2x80x9d is an integer equal to or greater than 3 but smaller than xe2x80x9cNxe2x80x9d and xe2x80x9cnxe2x80x9d of respective mixed specimens can be identical or different) or a set of mixed specimens and single specimens, making said specimen (mixed or single) in contact with a stationary phase which can bind with nucleic components of a specific structure in a container, and letting said stationary phase absorb and bind with said nucleic acids in the specimen, a second process which comprises a step of removing the other components which are not absorbed and bound from said stationary phase, and a third process which comprises the steps of applying a washing liquid to said stationary phase, discharging said washing liquid, and thus washing said stationary phase on each of the other mixed or single specimens,
letting said stationary phase absorb and bind with the target nucleic acids,
supplying an eluate into said container,
discharging the eluate from the stationary phase, and
thus eluting the nucleic components from said stationary phase.
A recovery apparatus which is another embodiment of the present invention comprising the steps of making a specimen containing nucleic components in contact with a stationary phase which can bind with said nucleic components in an area, causing said stationary phase to absorb and bind with said nucleic components, and eluting said nucleic components for recovery comprises a means which scans over said in a preset sequence, a first means which supplies said specimen to said area, a second means which removes the other components which are not absorbed and bound from said stationary phase, and a third process which comprises the steps of applying a washing liquid to said stationary phase, discharging said washing liquid, and thus washing said stationary phase on each of the other mixed or single specimens, a third means which supplies a washing liquid to said area, and a mean which supplies an eluting solution to said stationary phase, wherein said first, second, and third means exert upon each specimen in sequence to cause said stationary phase to absorb and bind with nucleic acids and the eluting solution is supplied to said stationary phase to elute said nucleic acids. Said apparatus can further comprise a means for mixing said specimen with a substance which accelerates nucleic components of a specified structure to be bound to said stationary phase prior to the step of adding said specimen to said stationary phase to contact. Further, said area of said recovery apparatus is a capillary having a capillary tip at the end of the capillary. Said stationary phase can be put in said tip. Furthermore, said tip is mountable on and removable from said capillary.
Another embodiment of the present invention provides a recording medium storing a processing program which executes the steps of applying a specimen containing nucleic components to a stationary phase which can bind with said nucleic components, letting said nucleic components be absorbed and bound by said stationary phase, removing the other components which are left unbound and unabsorbed from said stationary phase, performing these two steps on each of the other specimens, letting nucleic components be absorbed by said stationary phase, supplying an eluting solution to said stationary phase, discharging the used eluting solution, and eluting said nucleic components from said stationary phase. This processing program can contain the steps of applying a washing solution to said stationary phase and removing the used washing solution after a step of removing the other components which are left unbound and unabsorbed from said stationary phase.
By repeating absorption, binding, and washing on the identical stationary phase and finally eluting nucleic components from said stationary phase, the method of the present invention can recover nucleic components without reducing the concentrations of nucleic acids and consequently, this method enables detection of target nucleic acids such as target viruses.
For example, to inspect 50 specimens, said method can mix up all of the specimens or two or more specimens as unit samples (a mixed sample or a set of mixed samples and single samples), perform absorption, binding, washing, and elution on each of the sample unit on a single stationary phase. This method is applicable even when the concentration of nucleic acids of a specimen is very low to be detected. This method can efficiently recover nucleic acids without reducing the concentration of nucleic acids. When a specimen contains an extremely low concentration of a specific nucleic acid to be detected, it is preferable to inspect the specimen alone without mixing it with the others.